Discovery and evaluation of pyrimidine-2,4-dione derivatives as novel SARS-CoV-2 Mpro inhibitors with antiviral effect.

The COVID-19 pandemic has underscored the persistent threat of zoonotic coronavirus transmission to global health. In this study, we targeted the clinically validated SARS-CoV-2 main protease (Mpro) and implemented a scaffold-hopping strategy to design and synthesize 32 pyrimidine-2,4-dione derivatives, aiming to explore underutilized interactions within the S1, S1', and S2 subpockets. Structure-activity relationship (SAR) analysis identified compound 17 as a potent Mpro inhibitor (IC50 = 21.1 nM) with outstanding antiviral activity against the SARS-CoV-2 JN.1 variant (EC50 < 2 nM). Furthermore, X-ray crystallography of the Mpro-compound 15 complex unveiled a previously unreported T-shaped π-π interaction between the P1'-phenyl ring and His41. These results demonstrate the effectiveness of structure-based optimization of the pyrimidine-2,4-dione scaffold for the development of novel coronavirus Mpro inhibitors.